Themis J. Michailides received his M.S. degree in irrigations from the University of Athens, Greece, and his M.S. and Ph. D. degrees from the University of California –Davis. He joined the faculty of the Univ of California-Berkeley for three years before becoming a faculty member of the Univ of Calif. Davis where he is currently a professor and plant pathologist located at the Kearney Agric. AF36 in Almonds – Another Grower Tool for Aflatoxin Control in the Orchard
Themis Michailides
Current Cooperators: Ramon Jaime-Garcia, Teresa M. Garcia- Lopez, and John Lake
& Dr. Peter Cotty USDA/ARS & University of Arizona, Tucson, AZ AF36 Registration in Feb 2012
• Acreage: 330,000 acres • In production: 250,000 acres • Production: 690 million lbs. (= 313,500 tons of in shell pistachios)
AF36 Registration in Aug 2017
• Acreage: 1,110,000 acres • In production: 900,000 acres • Production: 2,050 million lbs. (= 930,000 tons of kernels)
AF36 Registration in Aug 2017
• Acreage: 7,500 acres • In production: 7,000 acres • Production: 21,000 lbs (= 9,560 tons of dried figs) Molds that can produce aflatoxin in almond (also in pistachio and fig) orchards in California
Aspergillus flavus Aspergillus parasiticus Aspergillus flavus and A. parasiticus produce:
Aflatoxins B1, B2, G1, G2, M1
O O
2 O 1 3 4 H 12 B1 is the most potent 15 6 5 7 14 11 aflatoxin; it can 13 8 cause liver cancer 16 O O 10 OCH H 9 3
Aflatoxin B1 Frequency and tolerance limits of aflatoxin contamination in California almonds
Frequency: 1 nut in 32,000 nuts
Regulatory limits for aflatoxins • USA Aflatoxin B110 ppb Total aflatoxins 15 ppb (in almonds for direct • European Union consumption) Aflatoxin B1 8 ppb Total aflatoxins10 ppb
… in milk: 0.5 ppb
In almonds “further processed”: 12 ppb B1; & 15 total aflatoxin Nut samples contaminated with aflatoxins (data of Dried Fruit Association in Fresno from 1985-1989 aflatoxin analyses) Aflatoxin contamination of almonds
Preharvest vs. Postharvest? (if almond nuts are dried quickly after harvest, stored properly, and kept dried). Preharvest problem!
Leaky stockpiles; no proper storage, etc… Postharvest Life cycle of Aspergillus flavus in almond orchards
navel orangeworm mummies
conidia in the air
navel Sclerotia in orangeworm soil and mummies Survival on orchard sclerotia in debris or on soil Relationship of navel orangeworm infestation and aflatoxin levels 40 > 0.0 ppb > 10 ppb 30 > 100 ppb
20
10
Samples with aflatoxin (%) 0 0-0.5 0.5-1.0 1.0-2.0 >2.0 Navel orangeworm (%) Kings County 2016: Regression of Average Weekly NOW damage over time (Day 7 = August 28)
0.08 y = 0.005e0.0489x Source: Dr. Joel Siegel, ARS-USDA R² = 0.94867 0.07
0.06
0.05
0.04
0.03 Percent NOW Damage NOW Percent
0.02
0.01
In0 general, the damage of NOW you have after the first week of harvest7 will be14 doubled21 following28 the third35 week of42 harvest (according49 56 toDays Dr. Joelfrom Siegel) August 21 Association of NOW with aflatoxigenic fungi
A. flavus
Si10
Sticky traps with NOW moths Effect of feeding sites (wounds) in almond on levels of aflatoxin contamination Sanitation
Sanitation
Sanitation Aspergillus flavus in almond orchards
S strain L strain (small sclerotia): almost all (large sclerotia): 50%:50% toxigenic toxigenic:atoxigenic Delivery of AF36 inoculum for treatment of pistachio orchards in 2012 AF36 Inoculum Application rate: 10 lbs. per acre
AF36 inoculum As applied
Irrigation is needed for spore production Sporulation
After growth of AF36 Sorghum is now used as a carrier of AF36 atoxigenic strain After irrigation, the wet seeds will produce spores of AF36 Non-treated orchard
Tox Treated Orchard
Atox Atox Tox Atox Library samples for aflatoxin analysis
Samples taken at processing plant as nuts are being unloaded. ReductionReduction in contaminated in aflatoxin-contaminated samples withpistachio aflatoxin samples– second(1st and harvest 2nd harvests) 50 44.9% P value =0.0033
38.6% 39.9% 40 36.7%
30
20.4% 20
10
0 Reduction of contaminated samples (%) samples of contaminated Reduction 2008 2009 2010 2011 2008-2011 (Doster et al. (2014), Plant Disease 98:948-956) (4 years average) Reduction in aflatoxin-contaminated pistachio samples (2nd harvest) 100
85.4% 80
58.1% 60 54.6%
40
23.6%
20 No samples No 0 Reduction of contaminated samples (%) 2008 2009 2010 2011 2009-2011 (3 years average) Registration of Aspergillus flavus AF36 strain for use in pistachio in 2012
FEB 29, 2012
Since 2013, about 150,000 to 200,000 acres were treated yearly! Occurrence of A. flavus atoxigenic strains in almond-growing counties of California
Each colored circle represents a different atoxigenic strain; = AF36
Butte
Glenn Madera
Colusa Fresno
Tulare Merced
Kings
Kern
AF36 incidence: 3.0% to 8.5% Reduction of aflatoxigenic Aspergillus flavus/A. parasiticus in areas of an almond orchard treated with the AF36
100
80 A. flavus S strain A. parasiticus Nickels Soil Laboratory 60
40
no application
Percentage of isolates Percentage 20 AF36 AF36 AF36 AF36
0 June '07 Aug '07 July '08 Sept '08 Sept '09 Sept '10 June '11 Sept '11 June '12 Aug '12 nut samples Date Ability of AF36 to reduce aflatoxins when co-inoculated with highly toxigenic isolates
aflatoxin B 200 1 2A1L-11 : toxigenic isolate of A. flavus 150
4C1P-11 : toxigenic isolate 100 aflatoxin G1 of A. parasiticus
Aflatoxin (µg/g) Aflatoxin 50
0 2A1L-11 2A1L-11 + 4C1P-11 4C1P-11 + 4C1P-11 4C1P-11 + AF36 AF36 AF36
Greater than 94% reduction in aflatoxins in comparison to levels in kernels inoculated with the toxigenic isolate alone Burkard spore trap in a pistachio orchard Registration of A. flavus AF36 Prevail
PISTACHIO, ALMOND, AND FIG: FOR USE ONLY IN THE STATES OF CALIFORNIA, ARIZONA, NEW MEXICO, AND TEXAS
LABELING ACCEPTABLE STATE OF CALIFORNIA DEPARTMENT OF PESTICIDE REGULATION Date: 08/07/2017 Reg. No. 71693-2-AA Conclusions of studies from the AF36 application in almonds
✓The AF36 persists well in the soil.
✓It does not cause any increase in nut decay.
✓Results on almond were similar to pistachio results.
✓The atoxigenic strain AF36 Prevail® is registered now and can be applied in 2018.
Rate: 10 lbs per acre Application timing: Late May to early/mid-July 40 lbs capacity
Some Challenges…
✓ Soil moisture (and temperature) ✓ Timing of application ✓ Harvest time ✓ Insect seed pests ✓ Predators Effect of the soil moisture on sporulation of Aspergillus flavus (AF36 Prevail®) on grains of product
45
36
27
18
9
0
Sporulation A. (AF36) flavus Sporulation <6 6-12 13-18 19-25 >25 Soil moisture (%)
Inoculum dropped into a very wet soil will not produce any spores of AF36; it will decay Warmer temperatures favor sporulation of the AF36 fungal strain on the AF36 product
)
6 200
59°F 68°F 95ºF 77°F 150 86°F 95°F
86ºF 100
50 77ºF
Spores of AF36 produced / wheatSpores seed AF36 (x10 of 0 2 3 4 5 6 7 Time (days) Effect of date of application on sporulation of Aspergillus flavus strain AF36
100
80
5/23/13 60 6/10/13 6/27/13 40 7/15/13 AF36 (%)AF36 Control
20
0 2013 2014 2015 Sample Date (year) Best results with the 15 July application Harvest and stockpiling
Hulls moisture <12%; kernel moisture 6% Predation of grain by insects
Ants Predation of grain and decay by other fungi
Roly polies Fusarium spp. Suggestions for best AF36 application
✓The application method and product rate (10 lbs per acre) are the same as in other crops.
✓Apply product in late May to early / mid-July.
✓Irrigate within 3 days after application.
✓Make sure that most of the inoculum will be spread in the areas wetted by irrigation.
✓Avoid covering the inoculum by plowing or with too much water.
✓Do not spray herbicides 1 to 2 weeks after application.
✓Control the ants, other arthropods, and birds in the orchard.
<< This is a novel approach to reduce aflatoxin contamination >> Link of video on application of AF36: www.calpistachioresearch.org/training-videos/ Acknowledgments:
• Peter Cotty, USDA • Ramon Jaime-Garcia • Michael Braverman • Juan Moral • Joel Siegel, USDA • Teresa M. Garcia-Lopez • Jeff Palumbo, USDA • John Lake • Mark Doster • Peter Cotty, USDA • David Morgan • Ryan Puckett • Lorene Boeckler • Paulo Lichtemberg • Matthias Donner • Alejandro O. Beltran
USDA/ARS, IR-4 Project/Biopesticide Branch, Almond Board of California, Calif. Pistachio Research Board, Calif. Fig Institute, …
… and a multitude of growers